CHARACTERIZATION OF A GRANULE-INDEPENDENT LYTIC MECHANISM USED BY CTLHYBRIDOMAS

The mechanism(s) by wh ich CTL induce target cell lysis have not been clearly elucidated. Perforin and the cytotoxic cell proteinases (granz ymes) contained within the granules of CTL and NK, have been implicate d, but abundant evidence for the existence of alternate lytic pathways has accumulated. In this report we characterize the mechanism of kill ing used by two cytolytic hybridomas (PMM-1 and MD90) that express nei ther perforin nor the granzymes. These characteristics are compared wi th results obtained by using a representative Ag-dependent, granule-co ntaining T cell clone in cytolysis assays. The major differences were that the granule-negative hybridomas could lyse a variety of target ce lls in the presence of cyclosporin and the absence of calcium. All the effecters could kill in the presence of protein synthesis inhibitors (cycloheximide and emetine) and induced DNA fragmentation in the targe t cells. The cytolytic hybridomas had to be stimulated to be cytolytic and this activation required the presence of calcium, was dependent o n protein synthesis, and inhibited by the addition of cyclosporin. Alt hough TNF was shown not to be involved, the sensitivity of the target cells to lysis by the granule-negative killers correlated with the lev el of expression of Fas Ag. With the use of L1210 and an L1210 cell li ne transfected with Fas cDNA we demonstrated that these MD90 and PMM-1 kill the latter much more effectively and that this increase was effe ctively inhibited with anti-Fas Ab. Furthermore the lack of sensitivit y to cyclosporin, cycloheximide, emetine, and EGTA was confirmed with these targets. We conclude that these two cytolytic hybridomas use the Fas lytic pathway to induce lysis in target cells.